Immunochemical determination of substances contained on textile fibers or polymers

ABSTRACT

A method for the detection of substances contained on textile fibers or other natural or synthetic polymers is disclosed. The contained substances are identified with immunochemical methods, where the immunochemical reaction can occur on the fiber or polymer. The method is especially suitable for the determination of substances that can act as allergens.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of International Application No. PCT/EP97/06763filed Dec. 3, 1997, the disclosure of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is concerned with a method for the detection of substancescontained on textile fibers or other natural or synthetic polymers.

2. Description of Related Technology

Textiles or foods, e.g., luxury foods, have numerous substancescontained in them which could act as allergens for consumers, dependingon immunological disposition. These allergens trigger allergic reactionswhich are manifested with an extensive symptomatology, mostly by strongrelease of histamine. Erythema, irritation of mucosa, asthmaticreactions, or even drop in blood pressure with shock-like states mayresult in allergic persons. Therefore, it is of great importance forallergic persons to identify substances that represent a risk potentialaccording to the particular individual allergic profile in textiles andfoods, e.g., luxury foods, etc., with a rapid process that is easy toperform.

U.S. Pat. No. 5,324,642 describes a method for the analysis of ananalyte in a keratin structure (e.g., hair, nails), in which an enzymeand a compound with low redox potential are allowed to act on a sampleof the keratin structure, in order to degrade the structure and todissolve the analyte in this digesting solution. The detection of theanalyte is preferably done by immunoassay techniques on a protein basis(that is, with antibodies).

In Dewair et al., J. Allergy Clin. Immunol. 76 (4), 537-542 (1985), “Useof immunoblot technique for detection of human IgE and IgG antibodies toindividual silk proteins”, silk proteins are extracted from silk,separated with the aid of polyacrylarnide gel electrophoresis (that is,not with antibodies) and incubated with human serum which may containIgE- and IgG-antibodies against silk proteins. The detection of theantibodies bound to the silk proteins is carried out using theimmunoblot technique.

In B. M. Hausen et al., Deutsche Medizinische Wochenschrift, 109 (39),1469-1475 (1984), “Hosiery Dye Allergy”, it is reported that testpersons with hosiery allergy reacted allergically in the epicutaneoustest to isolated hosiery dyes (especially azo dyes) separated usingchromatography. A detection of the allergens with the antibodies is notdescribed.

SUMMARY OF THE INVENTION

The invention provides a method for detecting or quantitativelydetermining substances contained in textile fibers and/or other naturalor synthetic polymers with the aid of antibodies whereby a consumer cancheck the allergenic potential of a textile or of a food such as aluxury food before purchasing it.

According to the invention, a substance contained on a textile fiber orpolymer substrate is detected by directly contacting a surface of thesubstrate and the substance contained therein with antibodies thatspecifically bind to the substance, and detecting or quantitativelydetermining the antibodies bound to the substance.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be explained in more detail with the aid of thefollowing figures.

FIG. 1 shows schematically the detection of antibodies with the aid offluorescent-marked antibodies. For this purpose, irradiation with lightof a certain excitation wavelength is introduced. After excitation, thefluorescent markers emit a fluorescent light, which is filtered in orderto eliminate nonspecific signals. The fluorescent radiation of themarker molecule can be detected directly.

FIG. 2 shows schematically the binding reaction of a specific antibodyto an antigen on a textile fiber. In the case shown, this is afluorescent-marked antibody which is directed against azo dyes. Afterspecific binding of the antibody to the azo dye on the fiber, thedetection process can proceed.

FIG. 3 shows schematically the behavior of the antibodies which do notrecognize any epitope on the fiber. In this case, the fluorescencesignal of the antibody on the fiber is not produced.

DETAILED DESCRIPTION OF THE INVENTION

An advantage of the antibodies used for detection according to theinvention lies in the fact that they bind not only to dissolvedmolecules, but also to immobilized structures. This opens up theattractive possibility to determine substances that can act asallergens, directly on the fiber or the polymer. As a result, theexpensive desorption or cleavage of the contained substances, which alsoinvolves analytical uncertainties, can be eliminated. Therefore, in theinvention, the detection reaction is carried out directly on the fibersof a textile or on natural or synthetic polymers, and thus providesdirect detection of the potential allergens contained with the aid of adirect determination of the antibodies on the fibers or polymers.

Suitable fiber or polymer materials in which the contained substancescan be detected according to the invention include but are not limitedto cotton, wool or other animal hair such as horsehair, for example,silk, jute, sisal, hemp or flax and their converted products, forexample, viscose fibers, nitrate silk, and copper rayon e.g., REYONfibers, synthetic materials made of polyvinyl chloride, celluloseacetate, polycarbonates, polyamides, polyurethanes, polyimides,polybenzimidazoles, melamine resins, silicones, polyesters, polyethers,polystyrene, polymethyl methacrylate, polyethylene, polypropylene,polyvinyl acetate, polyacrylonitrile, polybutadiene, polychlorobutadieneor polyisoprene and the copolymers of the monomers from which thesematerials are found, and combinations of the foregoing polymers.

It is preferred to use a nonspecific antibody to saturate the surface ofthe investigated substrate in order to minimize the nonspecific bindingof the specific, detecting antibody. Fibers or polymers possess manynonspecific binding possibilities for the detecting antibody, as aresult of which the actual detection reaction may be interfered with. Inorder to suppress this, it is favorable to saturate the surface of thematerial to be investigated with antibodies which are not directedagainst the substance to be detected. Since relatively large amounts ofthese antibodies are needed, they should originate, especially in thecase of polyclonal antibodies, preferably from rabbits, sheep, horses,cattle, goats or, most preferably, from sheep. The use of thenonspecific antibodies is typically done before the addition of thespecific antibody.

In an especially preferred embodiment, the nonspecific antibodiesoriginate from a different animal than the specific antibody. Specificand nonspecific antibodies differ also by their so-called “constantregion” F_(c). This permits a subsequent immunochemical reaction with asecondary antibody, which recognizes certain species-specific sectionsin the constant region of the specific detecting antibody. In theforegoing immunochemical detection of the specific antibody, naturallythis must not originate from the same animal species as the antibodyneeded for saturation.

Saturation with a nonspecific antibody is conceivable both in the caseof direct as well as in the case of indirect determination of thebinding of the specific antibody.

In another preferred embodiment, monoclonal or polyclonal antibodies orF_(ab) fragments thereof are used for the determination of the containedsubstances. For a very rapid determination, cost-effective polyclonalantibodies are preferred, while the monoclonal antibodies are especiallysuitable if it is desired to detect exactly-defined cross-reactivities,for example, in the sense of group selectivity. F_(ab) fragments are notsuitable for coupling with certain immunomarkers, but also they have acomparatively low tendency to bind nonspecifically.

When using antibodies in analysis, highly selective, simultaneouslystrongly binding antibodies are distinguished from weakly selective,simultaneously weakly binding antibodies (the corresponding antibodiescan be selected with the hybridoma technique). While a high selectivityof antibodies is ideal for the identification of a given substance—herea given substance contained, for example, an allergen—a not veryselective antibody has the advantage of considerable “cross-reactivity,”that is, it binds to similar structures with a similar binding constant.

The analytical sensitivity can be adjusted with the aid of the bindingstrength of the antibody. This is important for the practical use of atest because the degree of sensitivity needed depends on the givenrequirements.

In another preferred embodiment, the antibody is isolated byimmunostimulation of mammals, especially goats, sheep, pigs, rats,horses, rabbits, guinea pigs, poultry, cattle, and/or mice. This is doneby injecting into the mammal a certain, optionally allergenic componentor epitope of a certain, optionally allergenic component with sufficientantigenic properties. The mammal answers to this foreign antigen with animmune reaction, which leads to the formation of antibodies. In thisprocess, polygonal antibodies against the foreign antigen can beobtained.

A monoclonal antibody used according to the invention (for example, anantiallergen antibody) may be produced according to any suitable method.For example, spleen cells of immune-stimulated mice may be fused afterreaching the optimum antibody titer with in vitro raised myeloma cellsin polyethylene glycol and then raised on a HAT medium. In this medium,only the so-called hybridoma cells from spleen and myeloma can survive.In order to isolate suitable clones which produce the antibodies, theindividual clones are checked for their immunoglobulin production andthe producing clones are raised either preferably in vitro or,optionally, in mice. For further details on the production of monoclonalantibodies, reference is made to “Monoclonal Antibodies, Hybridomas: Anew dimension in biological analyses, Kennet et al., Plenum Press, NewYork, 1980”, “Antibodies, A Laboratory Manual, Harlow & Lane, ColdSpring Harbor Laboratory Press, 1988” and “Peters, F. H. , Baumgarten,H., Monoclonal Antibodies, 2nd Edition, Springer-Verlag,Berlin-Heidelberg-New York, 1990”, the respective disclosures of whichare incorporated herein by reference. The resulting monoclonalantibodies have the property of originating from one cell and thus allhave the same specific antibody properties, that is, they all recognizethe same epitope.

Antibodies according to the invention can be isolated and purifiedaccording to any suitable method (see Antibodies, A Laboratory Manual,Harlow & Lane, Cold Spring Harbor Laboratory Press, 1988), including butnot limited to the following methods: peptide and protein columnchromatography, HPLC, including “reverse phase” HPLC, protein isolationon protein A or protein G columns, and all conceivable combinations ofthese methods.

In a preferred embodiment, the antibodies for the detection reaction aremarked in such a way that their association with the allergen or foreignsubstance becomes visible, such as by marker molecules which serve tomark the specific detecting antibody. In this way, it is possible todetermine the presence of the antigen recognized by the antibodyqualitatively and/or quantitatively. This is possible in one way bydirect marking of the antibody directed against an optionally allergeniccontained substance (for example, by fluorescence marking, radioactivemarking, or covalent coupling to a suitable marker enzyme) orindirectly, through a second anti-anti-antigen-antibody directed againstthe specific anti-antigen-antibody, which can also be marked by anymethod described above or by any other suitable method. A method inwhich the detecting antibody (monoclonal, polyclonal, or F_(ab)fragment) or a secondary antibody directed against the detectingantibody is marked by the fluorescence method, is preferred. In thiscase, upon irradiation with a corresponding excitation wavelength, thedetection can be done directly on a visual basis.

The fluorescing molecule can be bound to the antibody used according tothe invention through primary valences or secondary valences. In thecorresponding optical excitation, the antibody then gives a fluorescencesignal and thus indicates the presence of the particular antigen.

Examples of suitable fluorescent probes includes1-anilino-8-naphthalenesulfonate, 1-dimethylaminonaphthalene-5-sulfonylchloride, fluorescein isothiocyanate, rhodamine and its derivatives anddonor-substituted oxindigo (Angew. Chem. 1996, 108, 1090-1093), whichare bound to the antibodies through primary or secondary valences.

As an alternative to fluorescence marking, in another preferredembodiment, marking of the anti-antigen-antibody is performed withradioactive isotopes. Preferred is the use of a γ-source, ¹²⁵I, withwhich a phenol group of a tyrosine of an antibody according to theinvention is marked. In this case, a scintigram would be prepared forthe detection of the antigen to be determined. However, the use of asoft β-source, such as ³H, ³⁵S or ¹⁴C, is possible.

Moreover, in another embodiment, the marker molecule can be an enzymewhich reacts with a substrate in a quantifiable manner. The enzymes arebound to the antibodies by covalent binds. Typically, alkalinephosphatase or peroxidase, especially horseradish peroxidase, which thencan be detected, for example, by oxidation with H₂O₂ in the presence oftetramethylbenzidine, whereupon a blue dye is formed, can be used, ordouble enzyme systems in which the product of the first enzyme reaction(of the enzyme that is covalently bound to the antibody), is furtherreacted in a second subsequent enzyme reaction and only the product ofthe second reaction is detected can be used.

Substances contained in fibers or polymers, which are proven to beantigens with the aid of antibodies may be considered allergens orforeign substances, include textile dyes, textile additives, and/ortheir accompanying substances and the fibers or polymers themselves. Inan especially preferred embodiment of the invention, therefore, theforegoing contained substances are detected with the aid of antibodies.

Although the foregoing substances are harmless to the majority of thepopulation, they represent a considerable health hazard to allergicpersons under certain circumstances. Since one cannot expect that,because of such a small group of affected persons these substances wouldbe prohibited, over the long term, a permanent need arises from this fora cost-effective analytical method which can be used by allergic personsin a routine manner when purchasing each new piece of clothing.

“Textile aids or their accompanying substances” are defined herein asthose substances which are needed during the textile processes (recoveryand manufacture of textile fibers, processing, finishing, andpackaging), and include but are not limited to the following examples:antistatic agents, antimicrobial substances, optical brighteners, agentsprotecting against insect damage, finishing agents, impregnating agents,stabilizers, stiffening agents, and agents for textile coating.

Dyes are also used in textile processes. They are generally usedtogether with dye solvents and dispersing agents. Dyes include reactive,direct, vat, sulfur, cationic coupling, and dispersion dyes.

Other contained substances that may be allergens or substances that areharmful to health in some other way, may be applied onto the fiberseither during the dyeing process or are used as textile aids. Examplesare pentachlorophenol (PCP), o-phenylenediamine, p-phenylenediamine,dimethyloldihydroxyethyleneurea, dimethylolethyleneurea,dimethyloltriazone, and dimethylolurea.

According to the invention, especially preferably dyes contained ontextile fibers or polymers are detected. Their detection with the aid ofan antibody and, preferably, fluorescence markers requires a number ofspecific boundary conditions, as set forth below:

Firstly, the excitation energy of the fluorescent marker is also emittedagain as fluorescent light, but is not transferred to the dye, and isthus lost (fluorescence quenching). An excitation wavelength of thefluorescent marker may be longer than the absorption wavelength of thedyes to be detected, so that energy transfer is not possible. However,such a method would greatly limit the number of detectable color tonesand would completely fail in the case of turquoise-colored dyes, becausethese absorb at the long-wavelength limit of the visible region so thata fluorescent marker that would be suitable for this would have tofluoresce not in the visible but in the NIR region, requiring detectionwith a machine. A suitable method is to position the fluorescence markerat a sufficient distance from the dye chromophores, namely at a distancegreater than the Förster radius, which can be set as a minimum of 30 Åso that, as a consequence, no fluorescence quenching can occur by energytransfer. A sufficient distance would be given already if the dye is notadjacent to the binding arm of the antibody, but is in farther removedantibody structural regions. Suitable linking methods are available forthis.

A second, important point concerns the selection of a suitablefluorescence wavelength for the fluorescent antibody system. Thespectral region available for this is highly limited by the usualtreatment, for example, of textile fibers with optical brighteners(textile pretreatment and detergent additives), which fluoresce in theviolet-to-blue region so that a fluorescence maximum can be set atapproximately 435 nm. Since a sufficient safety distance would have tobe observed from the emissions of the brightener, the green spectralregion must also be avoided in these cases. Therefore, the fluorescentmarkers preferably fluoresce in the yellow region, the orange-redregion, or the red region.

Preferably, however, the long-wavelength red spectral region is also tobe avoided because the sensitivity of the human eye or that ofphotomultipliers decreases greatly at these wavelengths. Therefore,especially preferably, the long-wavelength limit of fluorescence isbetween 650 nm and 700 nm.

For fluorescence immunodetection with a machine, spectral decompositionwould be a suitable method to separate the fluorescent antibody signalfrom the fluorescent background, which is caused by the opticalbrightener. For visual evaluation of the immunofluorescence, thefluorescent background produced by the optical brightener is alsodisturbing. This can be filtered out with a color filter with along-wavelength spectral edge (“edge filter”) at 450 to 500 nm. However,for a simple routine analytical method, observation through a filter,preferably through a yellow plastic film is sufficient.

Should the detection of the immunofluorescence take place with highsensitivity, then all fluorescent background of the fibers or polymers,caused by the production or by impurities, will disturb the process.This problem can essentially be eliminated by the use of fluorescentmarkers with an increased Stokes shift. Such an increase of the Stokesshift follows from special structural requirements. These requirementsare generally not fulfilled by occasional available substances becauseusually a normal Stokes shift is found therein. The fluorescence of themarkers can be separated with the usual optical filters from thefluorescence of the background, as is known in the art.

In one preferred embodiment, among the textile dyes, dispersion dyeswhich typically have a potentially allergenic character are detectedaccording to the method. Dyes that may be detected according to theinvention include but are not limited to the following: DISPERSION-BLUE124, DISPERSION-BLUE 3, DISPERSION-BLUE 7, DISPERSION-YELLOW 3,DISPERSION-YELLOW 9, DISPERSION-ORANGE 3, DISPERSION-ORANGE 76,DISPERSION-RED 1, and metholmelamine.

In another preferred embodiment, among textile dyes, dyes with an azogroup (“azo dyes”), which can also have a potentially allergeniccharacter, are detected.

Moreover, the analysis of azo textile dyes has assumed specialimportance because of the prohibition of certain such dyes bygovernmental agencies (e.g., the 2nd to 4th Amendment of the CommoditiesRegulation). Since the prohibition may be extended to additional azodyes, generally there is a high demand for analytical capacity for theidentification of such dyes. Firstly, for a detection method, it isnecessary that it allows the identification of certain azo dyes incolored textiles and, on the other hand, methods are needed with whichthe azo group can be directly detected as a substructure. The latter isof special importance because more than 10,000 different azo dyes areknown. The development and application of special methods ofdetermination for each of these azo dyes would mean an extraordinarilyhigh technical demand. Another aggravating factor is that not only arenew azo dyes designed and synthesized without any problem, but inindustry, frequently azo dyes are also used as reactive dyes, which arebound covalently to the fiber and therefore cannot be extracted orotherwise removed. Identification of such fiber-dye conjugates requiresbreaking of chemical binds for conventional methods of determination,which would bring about additional uncertainties in the analyticalmethod.

The development of a universal analytical method for the identificationof azo dyes is therefore highly desirable. Thus, methods are preferredin which the antibodies show a high selectivity toward partial molecularstructures of azo dyes. According to the invention, this is possible bythe use of antibodies against the inherent azo group of azo dyes as anantigenic substance. Such antibodies that are used according to themethod thus represent universal reagents for azo dyes.

The sensitivity for the detection of the azo group can be adjusted withthe binding strength of the antibody. This is important for thepractical use of a testing system because different degrees ofsensitivity are necessary depending on the requirements. Then, the classof azo dyes can be detected via cross-reactivities using one or a fewantibodies. However, depending on the requirements, it is favorable touse either only one antibody and take into consideration differences inthe cross-reactivities, or to level out the differences by the use ofseveral antibodies, which take into account the specific, slightlydifferent, binding relationships around the azo group.

In another preferred embodiment, antibodies are used which, in additionto the azo group, require other molecular parts as a component of therecognition structure, the epitope. These additional antigenic molecularstructures are then characteristic for individual azo dyes or forsubgroups of azo dyes. Thus, certain azo dyes can be identified anddetermined quantitatively with such antibodies.

In an especially preferred embodiment, for this purpose, the antibodiesare coupled with a fluorescent marker through primary or secondaryvalences. Preferably, these are markers with a yellow, orange-red, orred emission signal. The fluorescent markers can be bound directly tothe detecting antibody or to a secondary antibody which is directedagainst the detecting antibody. In order to produce a large distancebetween the chromophore and fluorescent marker, the marker is in aregion which is away from the paratope of the antibody.

The immunochemical detection method of azo dyes on textile fibersaccording to the invention is necessary because certain azo dyes are ona prohibition list. Moreover, the methods according to the invention canalso be used very generally for the detection of substances that giverise to concern about health, in fibers or polymers, especially intextiles. However, the methods according to the invention can also beused for other problems, such as for the identification of certainsubstances contained in polymers or fibers for the detection of theirorigin. Application of the method according to the invention in thecriminological area is also possible.

We claim:
 1. A method for detecting a presence of a substance contained in a substrate, which substrate is selected from the group consisting of textile fibers, natural polymers, and synthetic polymers, said method comprising the steps of: (a) directly contacting the substrate and the substance therein with first, specific antibodies that specifically bind to said substance; and, (b) detecting or quantitatively determining the first antibodies bound to said substance as an indication of the presence of said substance.
 2. The method of claim 1 wherein prior to step (a) the substrate surface is saturated with second, non-specific antibodies which do not specifically bind to an epitope of the substance to be detected.
 3. The method of claim 2 wherein the nonspecific antibodies originate from a species different from that from which the specific antibodies originate.
 4. The method of claim 1 wherein said specific antibodies are selected from the group consisting of polyclonal antibodies, monoclonal antibodies, F_(ab) fragments derived from polyclonal antibodies, and F_(ab) fragments derived from monoclonal antibodies.
 5. The method of claim 1 wherein said specific antibodies are obtained from an animal selected from the group consisting of mice, rats, rabbits, guinea pigs, sheep, goats, cattle, horses, and poultry.
 6. The method of claim 1 wherein said specific antibody is marked with a marker selected from the group consisting of radiochemical markers, enzymatic markers, and fluorescence markers.
 7. The method of claim 1 wherein the substance to be detected with antibodies is an antigen selected from the group consisting of textile dyes, textile aids, byproducts of textile dyes, byproducts of textile aids, and components of the substrate.
 8. The method of claim 1 wherein the substance to be detected is a dispersion dye.
 9. The method of claim 1 wherein the substance to be detected is an azo dye.
 10. The method of claim 9 wherein the antibody is specific for the azo moiety of the dye molecule but not for other characteristic parts of the dye molecule.
 11. The method of claim 9 wherein the specific antibody is specific to the azo moiety of the dye molecule and to at least one other characteristic part of the dye molecule.
 12. The method of claim 1 wherein a fluorescence marker with yellow fluorescence, orange-red fluorescence, or red fluorescence is coupled through primary or secondary valences to a structural region of the specific antibody which is distal from a paratope of the specific antibody.
 13. The method of claim 1 wherein said substrate comprises a material selected from the group consisting of cotton, animal hair, silk, jute, sisal, hemp, flax, and converted products thereof.
 14. The method of claim 13 wherein said substrate is selected from the group consisting of wool, horsehair, viscose fibers, nitrate silk, and copper rayon.
 15. The method of claim 1 wherein said substrate comprises a synthetic material selected from the group consisting of polyvinyl chloride, cellulose acetate, polycarbonates, polyamides, polyurethanes, polyimides, polybenzimidazoles, melamine resins, silicones, polyesters, polyethers, polystyrene polymethyl methacrylate, polyethylene, polypropylene, polyvinyl acetate, polyacrylonitrile, polybutadiene, polychlorobutadiene, polyisoprene, and copolymers thereof. 